6-methyl-delta5,7 sterols and methods to prepare same



3,468,875 6-METHYL-A STEROLS AND METHODS TO EREPARE SAME Bjarte Liiken, Shrewsbury, and Marcel Gut, Worcester,

Mass., assignors to Phytogen Products, Inc., Mamaronecir, N.Y., a corporation of Delaware No Drawing. Filed Nov. 26, 1965, Ser. No. 510,075 Int. Cl. C07c 169/60, 171/00, 167/26 US. Cl. 2-60239.55 13 Claims ABSTRACT OF THE DISCLOSURE The present invention relates to the preparation of 6- methylA sterols.

AcOkk/ Per-acid nited States Patent 0 W N l 3,468,875 Patented Sept. 23, 1969 ice ing the 6-methyl A sterols of the present invention. Thus, yeast (ergosterol), whale liver, halibut, and tuna liver, cattle spinal cords, the residue remaining from the vacuum distillation of herring oil fatty acids (cholesterol), the unsaponifiable material present in vegetable oils from soya beans, safilower seeds, peanuts, and cotton seeds (stigmasterol and sitosterol). These naturally occurring sterols are all closely related as can be seen from their chemical terminology, ergosterol being 3fl-hydroxy- 24B-methyl cholesta-5,7,22-triene; stigmasterol being 3/8- hydroXy-245-ethy1 cholesta-5,22-diene; and sitosterol being 3,8-hydroxy-24-ethy1 cholesta-S-ene.

Ergosterol already contains the conjugated 5-6 and 78 double bond system desired for practice of this invention. The other sterols named above can, however, be converted from A to the A configuration by techniques known to the art, these techniques per se not forming part of the present invention. To repeat, practice of the present invention employs at A sterol as the starting material.

The compounds in question and the general procedure are described below with reference to the following for- .mula sequence in which ergosterol acetate is employed as the starting material:

h R (l Pyridine P 'd'n yr! 1 e AcO H( (EH:

The first step (1) in the synthesis invloves the selective epoxidation of the 5,6 double bond of the A sterol. The direct epoxidation is effected by reaction of the sterol (A) with an organic per-acid such as perbenzoic acid, preferably meta-chloroperbenzoic acid. Peracetic acid, permaleic acid, and monoperphthalic acid also may he employed. It has, however, been found that employment of the organic per-acid as such leads to undesired side reactions, primarily the 5,6-epoxide forms, but reacts further with the carboxylic acid which is formed by reduction of the appropriate per-acid used, giving rise to opening of the epoxide to form the 6-ester of a S-hydroxysterol, a compound which is not considered desirable. Thus the use of not neutralized per-acids such as perbenzoic, peracetic, m-chloroperbenzoic acids would lead to the formation of 6-benzoyloxy, 6-acetoxy and 6-metachlorobenzoyloxy esters, respectively. Also, the organic per-acid adds to other double bonds present in the sterol molecule and in particular attacks the side chain double bond in ergosterol and stigmasterol.

These side reactions can be avoided by first neutralizing the per-acid, e.g., with aqueous caustic and then contacting the per-acid salt with the A sterol in the presence of a water immiscible solvent. Under such conditions, good selective results are obtained, i.e., the Set, 6a-epoxide forms in high yields; other double bonds in the sterol remain unattacked. In practice, the 50:, 6a-epoxides of both ergosterol and 7-dehydrostigmasterol are formed in good yields as are the epoxides of 7-dehydrochloresterol and 7-dehydrositosterol.

Metachloroperbenzoic acid is a preferred per-acid. The other per-acids named above worked quite well, but are not as convenient because of a lower peroxide assay and because preparation in situ causes some difliculties with exact neutralization. On the other hand the m-chloroperbenzoic acid is a crystalline powder which can be easily weighed and assayed by oxidometric titration. It is available in an assay of 85%, balance being assumed for neutralization purposes, to consist of the corresponding metachlorobenzoic acid. An additional advantage of the metachloroperbenzoic acid is that only the 5a,6a-epimeric epoxide is formed, which is the desired one.

Following the epoxidation (step 1) the 50,6ct-6POXld6 (Formula B) is subjected to a Grignard alkylation employing methylmagnesium bromide (step 2) to effect a C opening of the epoxide. An argon atmosphere above the reactants seems to be more eifective than a nitrogen atmosphere or absence of an inert gas cover.

Reintroduction of A into the sterol involves some problems of selectivity. Treatment with strong acids, a conventional technique for dehydrating steroids causes double bond migration from the desired A configuration to a A -diene, a reaction apparently accelerated by the presence of the 6-methyl substituent. Preferred practice of the present invention involves first acetylation of the 3-hydroxy substituent (step 3) in the presence of acetic anhydride and pyridine; then treating the resultant 3-acetate (Formula D) with a condensing agent, preferably thionyl chloride in a base (anhydrous pyridine). Other condensing agents such as phosphorous oxychloride in pyridine were satisfactory with, however, slightly lower yields.

The last step in the above formula sequence (step 5), i.e., saponification, is optional and may be omitted. Frequently the 3-acetate sterol compounds are more stable in air and light than are the corresponding 3-hydroxy derivatives. Indeed, saponification as shown for step 5 takes this relative stability into consideration through employment of mild conditions along with employment of a cover gas, e.g., nitrogen or argon.

The procedure illustrated by the above formula sequence can be modified to some extent. The epoxidation (step 1) can be effected on the 3-hydroxy sterol with almost the yield obtained from the 3-acetate. Similarly, the thionyl chloride dehydration (step 4) can be effected directly on the 3-hydroxy derivative rather than on the 3- acetate, with however some loss to a side reaction involving, apparently, chlorine substitution at the 3-position.

The above reaction sequence can be effected with 3- esters other than the 3-acetate. However, acetates at the 3-position are convenient and preferred because of their ease of formation and of hydrolysis. in addition the 3- acetates crystallize well, are convenient to handle, and are suitable for conversion of the provitamin into the D vitamin.

Upon ultra-violet light irradiation of provitamin sterols formed according to the practice of the present invention, the provitamin is converted to the corresponding vitamin according to the following formula:

(l) it It is well known in the art that relatively small changes in the side chain present in the various sterol molecules influence the antirachitic potency of the ultimate vitamin. Thus when the double bond in the ergosterol side chain is hydrogenated and the product irradiated, the D vitamin (e.g., D is more potent than D and is almost as active as D (from 7-deyhdrocholesterol). Irradiation of .7- dehydrostigmasterol (very similar to ergosterol) is known to produce a vitamin D of low activity.

However, introduction of a methyl group at the 7- position makes the A a tetra substituted double bond. and in all instances has been shown to enhance the vitamin D potency of the irradiated product over that of the precursor provitamin.

For further understanding of the practice of the present invention, the following specific examples are herewith presented.

Example I .tate in 30 ml. dichloromethane was added an ice cold solution of 9.75 ml. N/l aqueous sodium hydroxide diluted with 45 ml. water. The mixture was stirred in a flask, cooled in an ice water bath. As soon as possible after combining the 7-dehydrocholesterol acetate solution with the alkaline solution, addition of 1.68 g. of metachloroperbenzoic acid dissolved in 30 ml. dichloromethane was made over a period of two minutes. Stirring in the ice water bath was continued for a period of 10 minutes after completion of the addition. The mixture was then separated in a separatory funnel and the dichloromethane phase washed with 350 ml. of water. The dichloromethane was evaporated to dryness under reduced pressure. To the residue was added acetone and the evaporation to dryness in vacuo was repeated, and the residue was crystallized from acetone containing a little pentane. A total of 2.7 g. of crystals was harvested in three crops. After a recrystallization from acetone, an analytical sample was obtained of 3;8-acetoxy-5a,6ot-epoxycholest-7- ene, showing: M.P. 143l45; (cz) 8l (chlf.), calcd. for C H O C, 78.68; H, 10.47. Found: C, 78.95; H. 10.68.

Example II.35,5a-dihydroxy-6ot-methylcholest-7-ene To a solution of 23.5 ml. of 3 M methylmagnesium bromide in ether, diluted with 20 ml. tetrahydrofuran. was added slowly a solution of 2.97 g. 3p-acetoxy5a,6otepoxycholest-7-ene in ml. tetrahydrofuran. Prior to charging, the equipment was thoroughly flushed with argon (Linde 99.996% assay). An argon atmosphere was maintained during the addition, and the subsequent reflux. The mixture was refluxed for 20 hours after completion of the addition. Then the reaction mixture was allowed to cool while under argon. Slowly, drop by drop. 12 ml. of a saturated (at 25 C.) aqueous solution of ammonium chloride was added. During this addition vigorous magnetic stirring was maintained. Towards the end of the addition the opaque liquid separated to a clear supernatant and a white precipitate of the magnesium salts. The supernatant was decanted through a filter and the solids extracted with 3 portions of ether and the extract decanted through the same filter. The combined filtrates were concentrated to dryness under reduced pressure. The residue was dissolved in methanol and concentrated until initiation of the crystallization. The crystallizing mixture was cooled overnight and filtered the following morning.

2.47 g. of 3,8,5a-dihydroxy-6a-methy1cholest-7-ene was obtained in this manner, exhibiting M.P. 141-145 (chlf.); (L)D'-18 (chlf.), M (calcd.) 416.66, C H O Calcd.: C, 80.71; H, 11.61. Found: C, 80.67; H, 11.56.

Example HI.-5 a-hydroxy-3 B-acetoxy-6a-methylcholest- 7-ene To a solution of 2.07 g. of the 3,5-di0l from Example 11 in 8 ml. of dry pyridine was added 2 ml. of acetic anhydride and the mixture was left overnight at room temperture. The following day a couple of drops of Water were added to destroy the excess of acetic anhydride. One hour later the solution was poured into 3 liters of ice water with violent agitation. The microcrystalline precipitate was filtered, Washed with water, then with a little methanol. The analytical sample of 5a-hydroxy-35-acetoxy-6a-methylcholest-7-ene was obtained by recrystallization from methanol. M.P. 189-191; (M -55 (chlf.).

Example IV.3fi-acetoxy-6-methylcholest-5,7-diene To an ice cold solution of 3.5 g. of 5a-hydroxy-3B- acetoxy-6amethylcholest-7-ene in 42 ml. of dry pyridine was added slowly over a two-minute period an ice cold solution of freshly distilled thionyl chloride (1.4 ml.) in pyridine (28 ml. dry). The solution was agitated in an ice-Water bath for 30 mintues and then poured into one liter of ice water. The mixture was then heated allowing the compound to form an oily mass on the bottom and sides. Cooling caused the oil to solidify, permitting decantation of the water. The oily mass remaining in the beaker was crystallized from acetone. 1.75 g. of 6-methyl- 7-dehydrocholesterol acetate was obtained in this manner. The analytical sample was obtained by recrystallization from acetone. M.P. 110-115; (a) -44.9 (chlf.) M (calcd.) 440.68, C H O -Calcd.: C, 81.76; H, 10.98. Found: C, 81.81; H, 11.06.

Example V.3,8-hydroxy-6-methylcholest-5,7-diene To a suspension of 2.6 g. of the 6-methyl-7-dehydrocholesterol acetate (from Example IV) in 200 ml. of 90% aqueous methanol, was added 1 g. of anhydrous potassium carbonate. The mixture was refluxed for 1 hour, cooled, the crystals filtered and washed on the filter with methanol, then with water. The material was recrystallized from methylene chloride/methanol, and 1.6 g. of pure 6-methyl-7-dehydrocholesterol was obtained (plus additional material from the mother liquor). This material melted at about 135-138", and exhibited a specific optical rotation of (a) '-25 (chlfi). Ultraviolet measurements gave a curve having maxima at 5:273 m and e, 7940, and a shoulder at t=282 my. (e=7700), and an inflexion at )t, 292 to 302 mu.

M (C H O): 398.65. Calcd.: C, 84.35; H, 11.63. Found: C, 84.33; H, 11.89.

Example VI.3,B-acetoxy-5a,6orepoxy-24-methylcholesta- 7,22-diene To a solution of 1.59 g. of ergosterol acetate in 15 ml. dichloromethane was added an ice cold solution of 5.5 ml. N/ 1 aqueous sodium hydroxide solution diluted with 22.5 ml. water. The mixture was stirred in an ice water bath while a solution of 0.95 g. of m-chloroperbenzoic acid in ml. dichloromethane was added over a period of two mintues, and the stirring was maintained for an additional 10 minutes. The dichloromethane portion of the mixture was separated from the aqueous phase, and washed with 200' ml. of water.

The dichloromethane solvent was evaporated under reduced pressure. The residue was triturated with acetone and the resulting crystals filtered. One recrystallization from acetone-pentane provided the analytical sample showing: M.P. 192-194", (a) 59 (chlf.). The principal bands in the infrared spectrum were: A at: 3.4, 5.8, 6.1, 6.9, 7.35, 8.06, 10.3;1. Nuclear magnetic resonance spectrum showed 3 vinyl protones: Cq-'H at 324 c.p.s., and C H, C H at 308, 311, 312, and 315 c.p.s. (quartet:}=7 c.p.s.). It showed only one epoxide proton C ,-H at 177 and 181 (coupled}=4 c.p.s.). This proved that the double bond in the side chain remained unattached by the epoxidation reagent, and that the epoxide formed had the 5a,6a-configuration. The presence of the 7, 8 double bond is moreover confirmed by the absorption band at 6.1 1 in the infrared spectrum.

Example VIIa, b

The method of Example I was applied to the starting materials listed below and the corresponding 5a,6aepoxy derivatives obtained:

Starting material: Epoxide (a) 7-dehydrostigmasterol, 3fi-acetoxy-5w6u-epoxyacetate. 24ethyl-cholesta- 7,22-diene. (b) 7-dehydrositosterol, 3 B-acetoxy-S wfinc-epOXY- acetate. 24-ethyl-cholesta- 7-ene.

Example VIIIa-c By subjecting the starting materials as indicated below to the Grignard reaction with methylmagnesium bromide as described in Example II, the appropriate 6-methyl-5- hydroxy derivatives listed in the second column below Were isolated.

Starting material From Ex. End product (a) 3fl-acetoxy-fiafia-epoxy- II 3 9, 5a-dihydroxy-6 24- 24-methylcholesta-7,22- dimethyleholesta-7,22- diene. diene.

(b) 35-acetoxy-5a,6a-epoxy- VIIa 3B,fia-dihydroxy-tia-methyl- 24-ethylcholesta7,22-diene. 24-ethy1cholesta-7,22-diene The yields obtained in all three cases were in the order of -85% of theory.

Example L(ac Starting material: 5,7-diene obtained 313,5a dihydroxy 611,245 di- 6-methylergosterol,

methylchloesta-7,22-dione. acetate M.P.

35,50 dihydroxy 6a methyl- 6-methyl-7-dehydro- 24-ethylchloesta-7,22-diene. stigmasterol,

acetate.

3,8,50: dihydroxy 60c methyl- 6-methyl-7-dehy- 24-ethylchloest-7-ene. drositosterol,

acetate.

As expected, the infrared spectra of these compounds were almost identical with the 6-methyl-7-dehydrocholesterol acetate of Example IV. The ergosterol and stigmasterol derivative showed a characteristic band at 103 which the sitosterol and cholesterol derivative did not have, and which is indicative of the trans-disubstituted double bond in the aliphatic side chain of these two compounds (A Example Xa-c The acetates from the previous Example lXa-c are saponified according to the conditions described in detail in Example IV.

Starting material: 5,7-diene sterols obtained 6-methyl ergosterol, 6-methylergosterol M.P. about:

acetate. 145-150 (M -50 (chlf.).

6-methyl-7-dehydro- 6 methyl 7 dehydrostigmastigmasterol, acesterol M.P. about: 137-143 tate. (ab-21 (chlf.).

The ultraviolet spectrum of the 5,7-diene sterols above were in all pertinent details identical with that described for the 6-methyl-7-dehydrocholesterol (Example V).

What is claimed is:

1. A compound of the formula:

wherein A is selected from the group consisting of single and double bonds; R is selected from the group consisting of H, CH z s;

B is selected from the group consisting of H on R is selected from the group consisting of H and acetate.

2. A compound of the formula:

wherein A is selected from the group consisting of single and double bonds;

R is selected from the group consisting of CH C H 3. 3,6-hydroxy-6-rnethylcholest-5,7-diene.

4. 6-methyl ergosterol.

5. A compound according to the formula of claim 2,

as follows:

6. A- compound according to the formula of claim as follows: 0

7. A compound according to the formula of claim 2 as follows:

Y? /(:i CH3 AGOQIJ l CH 8. A compound according to the formula of claim 2 as follows:

10. The process of epoxidizing A sterol selectively to the A", 5a,6tx-epoxide thereof which comprises subjecting the sterol or its 3-ester to the action of an organic per-acid in the presence of a water immiscible solvent 9 for the per-acid and steroid and an aqueous solution of sufficient alkali hydroxide to neutralize the organic peracid.

11. The process of claim 10 wherein the epoxide is then converted by reaction with methyl magnesium bromide to the A S-OH, 6-Me derivative thereof and thereafter by reaction with a condensation agent selected from the group consisting of thionyl chloride and phosphorous oxychloride in the presence of an organic base is converted to the A -6a-methyl derivative thereof.

12. The process of claim 10 wherein the organic peracid is m-chloroperbenzoic acid,

13. The process of claim 10 wherein the Z-acetate of the sterol is epoxidized,

References Cited UNITED STATES PATENTS 4/1965 Beal et al. 260-239.5

OTHER REFERENCES ELBERT L. ROBERTS, Primary Examiner US. (:1. X.R. 260-3972, 999 

